Although the vertebrate embryonic midline plays a critical role in determining the left/right asymmetric development of multiple organs, few genes expressed in the midline are known to function specifically in establishing laterality patterning. Here we show that a gene encoding protein disulfide isomerase P5 (PDI-P5) is expressed at high levels in the organizer and axial mesoderm and is required for establishing left/right asymmetries in the zebrafish embryo. pdi-p5 was discovered in a screen to detect genes down-regulated in the zebrafish midline mutant one-eyed pinhead and expressed predominantly in midline tissues of wild-type embryos. Depletion of the pdi-p5 product with morpholino antisense oligonucleotides results in loss of the asymmetric development of the heart, liver, pancreas, and gut. In addition, PDI-P5 depletion results in bilateral expression of all genes known to be expressed asymmetrically in the lateral plate mesoderm and the brain during embryogenesis. The laterality defects caused by pdi-p5 antisense treatment arise solely due to loss of the PDI-P5 protein, as they are reversed when treated embryos are supplied with an exogenous source of the PDI-P5 protein. Thus the spectrum of laterality defects resulting from depletion of the PDI-P5 protein fully recapitulates that resulting from loss of the midline. As loss of PDI-P5 does not appear to interfere with other aspects of midline development or function, we propose that PDI-P5 is specifically involved in the production of midline-derived signals required to establish left/right asymmetry.